A quantum dot is a semiconductor nanostructure that confines the motion of conduction band electrons, valence band holes, or excitons in all three spatial directions. Quantum dots (QDs) are traditionally chalcogenides (selenides or sulfides) of metals like cadmium or zinc (CdSe or ZnS), which range from 2 to 10 nanometers in diameter.
QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable simultaneous excitation of multiple fluorescence. Moreover, QDs are resistant to photo bleaching than organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity.
However, for in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. The toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, since emergent use of fluorescent QDs for molecular diagnostics and pathology is an important and clinically relevant application for semiconductor QDs. (Kairdolf. B. et al., Annual Rev. of Analytical Chem. Vol. 6: 143-162.)
In prevalent practice, the use of carbon nanoparticles in synthesis of quantum dots, have emerged as a new class of quantum dot-like fluorescent nanomaterials. Carbon nanoparticles are used since their particle size can be controlled between 3-20 nm. Carbon atoms linked in hexagonal shapes, wherein each carbon atom is covalently bonded to three other carbon atoms to form graphene sheets. Graphene has the same structure of carbon atoms linked in hexagonal shapes to form carbon nanotubes, but graphene is flat rather than cylindrical.
Graphene quantum dots (GQDs) are used as fluorophores for bioimaging, owing to their physicochemical properties including tunable photoluminescence, excellent photostability, and biocompatibility. GQDs usually less than 50 nm in size have been reported to have excellent fluorescent properties. Due to luminescence stability, nanosecond lifetime, biocompatibility, low toxicity, and high water solubility, GQDs are demonstrated to be excellent probes for high contrast bioimaging and bio sensing applications.
References may be made to prior art documents for methods of synthesizing GQDs using electrochemical processes, hydrothermal methods and the modified Hummers process for graphene oxide synthesis and cytotoxicity assays to determine the cellular uptake of the resultant GQDs formed by these processes.
US patent publication, US 2013/0175182 provides a process for the transformation of single walled, double walled or multi walled carbon nanotubes to nanoribbons composed of few layers of graphene by a two-step electrochemical process. The process involves oxidizing dispersed carbon nanotubes (CNT) to obtain CNT oxide and further reducing it to form graphene layers.
In research publication, Chem. Commun, 2011, 6858-6860, Zhu et al, describe a method of GQD preparation wherein modified Hummers method is used for graphene oxide synthesis and hydrothermal method for GQD synthesis to obtain GQDs of particle size of 5.3 nm. At concentrations of 2.6 mg/ml, cell viability of 80% is observed.
Further Jianhua Shen et al. in New J. Chem., 2012, 36, 97-101 reported one-pot hydrothermal reaction for preparation of graphene quantum dots surface-passivated by polyethylene glycol (GQDs-PEG) and their photoelectric conversion under near-infrared light, using small graphene oxide (GO) sheets and polyethylene glycol (PEG) as starting materials.
Juan Peng et al. (Nano Lett., 2012, 12 (2), pp 844-49) describes the acid treatment and chemical exfoliation of carbon fibers, to provide GQDs in the size range of 1-4 nm. The publication provides that the GQDs derived have no toxicity at concentrations of 0.05 mg/ml. However, the cytotoxicity of GQDs at higher levels is unaccounted.
Chang Ming Li et al., (J. Mater. Chem., 2012, 8764-66) provide a method to develop graphene quantum dots (GQDs) from XC-72 carbon black by chemical oxidation, however toxicity assays confirm maximum cell viability at concentrations of 0.1 mg/ml.
The toxicity of GQDs is attributed to their size, since small sized GQDs interact with various proteins and organelles inside the cell and disrupt cellular processes. Another reason for the toxicity is their ability to generate more reactive oxygen species (ROS). Polymers, especially PEG coating has been used in the literature to decrease the toxicity of GQDs. However, even after polymer coating the cell viability at higher concentrations (>1 mg/ml) is low. Probably because even though the ROS production is lowered by the polymer shell coating, the size of the GQDs after coating still remains small (sub 50 nm) and are still in the size range that can interact with intracellular proteins and organelles.
In the following research publications, references may be made to PEGylation of carbon nanoparticles and the cell viability determined at concentrations of 1 mg/ml or lesser than that.
Bhunia et al., (Scientific Reports, 2013, 3:1473) describe carbon nanoparticles (FCN) which are polymer coated with PEG and the dosage dependent cellular toxicity of these fluorescent nanoparticles. At 1 mg/ml concentration of the FCN-PEG composition, 55-60% cell viability is observed.
Zhuang Liu et al., (J. Am. Chem. Soc., 2008, 130 (33), pp 10876-10877) describe pegylated nano-graphene oxide (NGO-PEG) of size 5-50 nm for delivery of water insoluble cancer drugs produced by Hummers method.
Omid Akhavan et al., (J. Material. Chem., 2012, Vol. 22, 20626-33) describes nontoxic concentrations of pegylated graphene nanoribbons for selective cancer cell imaging and photothermal therapy. At concentrations of 1 mg/ml of the composition. 28% cell viability was obtained.
Further Lay C L et al. (Nanotechnology. 2010 Feb. 10; 21(6):065101) reports delivery of paclitaxel by physically loading onto poly (ethylene glycol) (PEG)-graft-carbon nanotubes for potent cancer therapeutics.
Toxicity assays of GQDs synthesized by methods of the above prior arts report minimum cell viability at GQDs concentrations of 1 mg/ml, and lesser than that, thus posing limitations in cellular imaging applications. However, to realize biomedical applications of GQDs, low toxicity of the GQDS at higher concentrations is desired for cellular imaging.
With a view to provide graphene quantum dots (GQDs) with decreased cytotoxicity levels at higher concentrations i.e. greater than 1 mg/ml, the present inventors have provided a biocompatible composition of one or more graphene quantum dots (GQDs) in a nanosized polymer matrix of polyethylene glycol which is larger compared to small sized GQDs as observed in the prior art. The PEG matrix aids in reducing the reactive oxygen radicals (ROS) generated by the GQD surface while keeping the small GQDs inside the matrix; thus, also reducing their undesirable interactions with cellular proteins and organelles.